Of all the macromolecules in living organisms, enzymes represent those which are the most complex in terms of structure and mechanistic properties. Enzymes are able to catalyze the transformation of all other biomolecules, providing the dynamics and very essence of life. Enzymes can aptly be considered natural bionanomachines which do chemistry. In particular, enzymes are proteins that accelerate the chemical transformation of a substrate molecule that binds to the active site of the enzyme in a thermodynamically and mechanically favorable manner, resulting in a chemical transformation of the substrate into a product molecule. Such enzyme catalyzed chemical transformations can include hydrolysis, oxidation/reduction, group transfer, isomerization, addition or removal of groups from double bonds, and ligation reactions. Enzymes catalyze reactions with high specificity and enormous rate accelerations, some having turnover numbers of millions of substrate molecules per second.
In the case of certain proteases, a catalytic triad is thought to be primarily responsible for the efficient hydrolysis (cleavage) of amide bonds in proteins and polypeptides, as well as ester bonds in certain biomolecule and synthetic substrates. For a serine protease such as Chymotrypsin, the catalytic triad motif is a close proximity arrangement of the serine (“Ser”) 195, the histidine (“His”) 57 and the aspartate (“Asp”) 102 amino acid residues in the polypeptide chain. In this catalytic triad, the serine hydroxyl group acts as a strong nucleophile, the histidine imidazole group as a general acid/base and the aspartate carboxyl group helps orient the histidine imidazole group and neutralize the charge that develops during the transition states. With the aid of this hydrogen bonding and exchange network within the reaction site, the catalytic triad functions as a reversible charge relay mechanism where protons are thought to be exchanged from one residue to another producing an efficient catalytic mechanism. While the stereochemical fit and binding between the substrate and enzyme is very important, it is the complex three dimensional (“3D”) protein structure which actually produces the dynamic mechanical properties in the catalytic triad that lead to efficient enzyme catalysis and turnover.
Most scientists who study enzymology are well aware that the remarkable catalytic properties of enzymes come from their complex 3D protein structure. Upon binding a substrate molecule, the enzyme carries out a rapid set of precise chemo-mechanical dynamic movements which converts the substrate(s) into the product molecule(s).
Over the past three decades a number of efforts have been made to create synthetic versions of enzymes which are sometimes called synzymes or enzyme mimics. Many of the synzymes are based on peptides, synthetic macromolecules and more recently nanostructures that are designed to closely resemble the active site of an enzyme. While these synthetic structures look similar to the enzyme active site they may not have the unique mechanical or dynamic catalytic properties to transform a substrate molecule into the desired product molecule in a repeated process i.e., turnover. Early work by one of the inventors of the present invention involved synthetic peptide structures which contain the same basic catalytic groups, a cysteine-sulfhydryl/thiol, a histidine-imidazole and an aspartate-carboxyl, that are in found the active site of Papain (Heller M J, Walder J A and Klotz I M, JACS, 99(8): 2780-2785, 1977). The synthetic peptide structures of that study were found not to exhibit any efficient catalytic properties, particularly with regard to turnover.
The natural enzyme Papain is a cysteine protease from the papaya plant, whose active site catalytic triad (Cys 25, His 159, and Asp 158) efficiently catalyzes the hydrolysis (cleavage) of both peptide (amide) bonds and ester bonds. Papain has a catalytic mechanism similar to Chymotypsin; the only difference is that a cysteine sulfhydryl/thiol group is the primary nucleophile in Papain. In Papain catalysis, the cysteine sulfhydryl/thiol group carries out a nucleophilic attack on the substrate amide/ester bond forming an acyl-cysteine intermediate. The histidine imidazole group is involved in the deacylation of the acyl-cysteine intermediate which leads to rapid turnover of the enzyme. In the case of the synthetic peptide structures which mimicked the Papain active site, acyl-group exchange was observed between the acyl-cysteine and the imidazole group however, back-attack by the cysteine sulfhydryl/thiol group prevented catalysis and any turnover in these synthetic peptide mimics. In this particular case, the back-attack is more formally an example of an intra-molecular acyl-transfer reaction between the cysteine sulfhydryl/thiol and the histidine imidazole, where the equilibrium greatly favors the reverse reaction for reforming the acyl-sulfhydryl/thiol group.
Other early work by one of the inventors of the present invention involved using synthetic DNA structures to catalyze the formation of peptide bonds (Walder et al., PNAS USA, 76 (1):51-55, 1979). This work demonstrated the potential for using amino acid modified DNA/RNA hybridizing structures and DNA templates to catalyze amide bond formation for peptide synthesis reactions. While the hybridized DNA/RNA structures provided very close proximity for the reacting groups, very little peptide bond formation was observed in the study.
In more recent work, systems and methods were developed wherein hydroxyl groups and imidazole groups were arranged in small synthetic structures (Roth et al., JACS 127: 325-330, 2005), as well as in nanostructured channels which assured their close proximity (Kisailus et al., PNAS USA, 103(15):5652-5657, 2006). These synthetic structures were designed to mimic the active site of Silicatein, a mineral-synthesizing enzyme that produces filamentous organic/inorganic cores of marine organisms, which utilizes both a serine hydroxyl group and histidine imidazole group for catalysis. Nevertheless, in these studies little or no turnover was observed in either the small synthetic structures or the precision nanostructures. Yet another example involving synthetic synzyme structures is disclosed in U.S. Pat. No. 6,048,690 to Heller et al., which describes the use of an electric field to enhance catalysis in a basic cysteine-histidine peptide immobilized on an electrode surface as a model for heterogeneous catalysis. However, no activity was observed, suggesting the basic peptide structures still require incorporation of other unique properties.
With regard to other enzyme mechanisms and their catalytic groups, some examples include: (1) Enolase, which catalyzes the conversion of 2-phosphoglycerate to phosphoenol-pyruvate uses a lysine amino group and a glutamate carboxyl group along with Mg2+ cations in the catalytic process; (2) Lysozyme, which catalyzes the hydrolysis of glycosidic C—O bonds in polysaccharides uses a glutamate carboxyl and an aspartate carboxyl in the catalytic process; (3) DNA polymerase, which catalyzes the synthesis of DNA uses three aspartate carboxyl groups, two Mg2+ cations and deoxynucleotide triphosphates (dNTPs) in the catalytic process; (4) Lactate Dehydrogenase, which catalyzes the reduction of pyruvate to lactate uses two arginine quanidinium groups, a histidine imidazole group and the reduced cofactor/coenzyme nicotinamide adenine dinucleotide (NADH) in the catalytic process; and (5) the water splitting/oxygen-evolving complex in plant photosynthesis utilizes tyrosine hydroxyl groups and four Mn2+ cations in this unique and highly important catalytic process. Thus, other catalytic groups which include glutamate carboxyl, the lysine amino, the arginine guanidinium and the tyrosine hydroxyl group; as well as metal cations (e.g., Mg, Mn, Ca) and various coenzymes/cofactors/prosthetic groups (e.g., NADH, FAD, ATP, dNTPs, Heme groups) are involved in enzyme catalysis. Such a diversity of catalytic groups is required in order to carry out the catalysis of a variety of other reactions including oxidation and reduction reactions; group transfer reactions; isomerization reactions; reactions involving the addition or removal of groups from double bonds; ligation reactions involving the formation of C—C, C—S, C—O, and C—N bonds by condensation reactions coupled to ATP or other energy rich molecules; and specialized reactions for photosynthetic driven water-splitting, oxygen evolution, and reductions including hydrogen production.